Cytokinins have been demonstrated to play a fundamental role in establishing seed size, decreasing tip kernel abortion and increasing seed set during unfavorable environmental conditions. The first naturally occurring cytokinin was purified in 1963 (Letham, D. S., Life Sci. 8:569–573 (1963)) from immature kernels of Zea mays and identified as 6-(4-hydroxy-3-methylbut-trans-2-enylamino) purine, more commonly known today as zeatin. In the main all naturally occurring cytokinins appear to be purine derivatives with a branched 5-carbon N6 substitutent. (See: McGaw, B. A., In: Plant Hormones and their Role in Plant Growth and Development, ed. P. J. Davies, Martinus Nijhoff Publ., Boston, 1987, Chap B3, Pgs. 76–93, the contents of which are incorporated by reference for purposes of background.) While some 25 different naturally occurring cytokinins have been identified, those regarded as particularly active are N6 (Δ2-isopentenyl) adenosine (iP), zeatin (Z), diHZ, benzyladenine (BAP) and their 9-ribosyl (and in the case of Z and diHZ, their O-glucosyl) derivatives. However, such activity is markedly reduced in the 7- and 9-glucosyl and 9-alanyl conjugates. These latter compounds may be reflective of deactivation or control mechanisms.
The metabolism of cytokinins in plants is complex. Multi-step biochemical pathways are known for the biosynthesis and degradation of cytokinins. At least two major routes of cytokinin biosynthesis are recognized. The first involves transfer RNA (tRNA) as an intermediate. The second involves de novo (direct) biosynthesis. In the first case, tRNAs are known to contain a variety of hypermodified bases (among them are certain cytokinins). These modifications are known to occur at the tRNA polymer level as a post-transcriptional modification. The branched 5-carbon N6 substituent is derived from mevalonic acid pyrophosphate, which undergoes decarboxylation, dehydration, and isomerization to yield Δ2-isopentenyl pyrophosphate (iPP). The latter condenses with the relevant adenosine residue in the tRNA. Further modifications are then possible. Ultimately the tRNAs are hydrolyzed to their component bases, thereby forming a pool of available free cytokinins.
Alternately, enzymes have been discovered that catalyze the formation of cytokinins de novo, i.e., without a tRNA intermediate. The ipt gene utilized in the practice of this invention is one such gene. The formation of free cytokinins is presumed to begin with [9R5′P] iP. This compound is rapidly and stereospecifically hydroxylated to give the zeatin derivatives from which any number of further metabolic events may ensue. Such events include but are not limited to (1) conjugation, incorporating ribosides, ribotides, glucosides, and amino acids; (2) hydrolysis; (3) reduction; and (4) oxidation. While each enzyme in these pathways is a candidate as an effector of cytokinin levels, enzymes associated with rate-limiting steps have particular utility in the practice of this invention.
One such enzyme is isopentenyl transferase (ipt). An isolated gene encoding ipt was described by van Larebeke et al., (Nature 252:169–170(1974)). Smigocki et al. (Proc. Nat'l. Acad. Sci. (USA) 85:5131–5135(1988)), employing the ipt gene from A. tumefaciens operably linked to either the 35S or NOS promoter, showed a generalized effect on shoot organogenesis and zeatin levels. Such unregulated production of cytokinins can result in unwanted pleiotropic effects. For example, with the constructions identified above, Smigocki et al. (supra) reported that typically complete inhibition of root formation was observed.
Attempts followed to express the ipt gene in a more controlled fashion. Medford et al. (The Plant Cell 1:403–413(1989)) reported placing the ipt gene under the control of a heat-inducible promoter and expressing same in transgenic rooted tobacco plants. While the levels of cytokinin rose dramatically following heat treatment, the promoters were not wholly satisfactory because the plants exhibited phenotypes associated with excess cytokinin levels even in the absence of thermal induction. See also: Schumulling, T. et al. (FEBS Letters 249(2):401–406(1989)). A more regulated response was reported in PCT Patent Application Publication No. WO91/01323, 7 Feb. 1991, and PCT Patent Application Publication No. WO93/07272, 15 Apr. 1993, both assigned on their face to Calgene, in which the ipt gene was fused to the chalcone synthase (chs) promoter from Antirrhinum majus and expressed in potato.
Additional ipt gene/promoter constructions have been reported. Smigocki et al., in U.S. Pat. No. 5,496,732, disclosed a gene construct capable of conferring enhanced insect resistance comprising a wound-inducible promoter fused to an ipt gene. Houck et al., in U.S. Pat. Nos. 4,943,674 and 5,177,307, disclosed several promoters (2AII, Z130 and Z70) coupled with genes encoding enzymes in the cytokinin metabolic pathway, in particular ipt for expression of such enzymes in tomato fruit. Amasino et al., in PCT Patent Application Publication WO96/29858 disclosed two senescence gene promoters operably linked to an ipt gene to inhibit leaf senescence in tobacco. See also: Gan, S. et al., (Science 270:1986–1988 (1995)). Roeckel, P. et al., (Transgenic Res. 6(2):133–141 (1997)) transformed canola and tobacco with an ipt gene under the control of a 2S albumin promoter from Agrobacterium. Increase in branching of inflorescences was noted, but increases in seed yield and seed weight were not observed.
There still exists a need for the controlled expression, both temporally and spatially, of cytokinin metabolic genes in plant seed and in those maternal tissues in which seed development takes place. This invention addresses this need by providing several useful genetic constructs and methods to modulate effective levels of cytokinin in plant seeds, developing plant seeds, and related maternal tissues. These related maternal tissues would include such tissues as the female floret, the ovary, aleurone, pedicel, and the pedicel-forming region. The maternal tissues are also referred to as “grain initials” or “seed initials”.
This invention differs from the foregoing approaches in that it provides tools and reagents that allow the skilled artisan, by the application of, inter alia, transgenic methodologies to influence the metabolic flux in respect to the cytokinin metabolic pathway in seed. This influence may be either anabolic or catabolic, by which is meant the influence may act to increase the flow resulting from the biosynthesis of cytokinin and/or decrease the degradation (i.e., catabolism of cytokinins). A combination of both approaches is also contemplated by this invention.